Bone Marrow Transplantation (2013) 48, 162–167 & 2013 Macmillan Publishers Limited All rights reserved 0268-3369/13 www.nature.com/bmt

REVIEW Diagnosis of acquired aplastic anemia

A Rovo´ 1, A Tichelli1 and C Dufour2, on behalf of the SAA-WP EBMT

Since the introduction of the concept of aplastic anemia (AA) by Paul Ehrlich in 1888 and despite the current better understanding of the underlying mechanisms involved in this disease, a clear delimitation among BM failure syndromes is still a matter of debate. The diagnosis of AA can be difficult basically due to the overlapping morphological characteristics with other BM failure disorders. This paper reviews critical data relevant to the diagnosis of acquired AA and recommends work out steps and main considerations to determine severity and characterization of the disease. The diagnostic challenge in the differentiation between AA and hypoplastic myelodysplastic syndromes is also addressed. The definition of the response criteria to treatment belongs to the diagnostic tasks and it is included in this review as well as an overview of novel tools for the diagnosis of AA.

Bone Marrow Transplantation (2013) 48, 162–167; doi:10.1038/bmt.2012.230; published online 19 November 2012

The diagnosis of aplastic anemia (AA) is defined by the differential diagnosis with myeloproliferative/MDSs like juvenile coexistence of pancytopenia with persistent and unexplained myelomonocytic leukemia or other subtype of MDS.5,6 reduced marrow hematopoietic cellularity, with no major dysplas- Aspiration and BM biopsy provide the diagnostic clues. tic signs and fat cell replacement Radiotherapy and chemother- Although aspiration allows a better discrimination of cellular apy-induced aplasia do not belong to AA. For the diagnosis of AA, morphology, particularly by the assessment of dysplasia, trephine there are no specific markers and the diagnosis is reached by is crucial to assess overall cellularity, topography of hematopoietic exclusion of other reasonable entities. The diagnosis of AA can be cells and to exclude abnormal infiltrates by examining at least 4–5 difficult basically due to the overlapping morphological character- undistorted fields under  100 magnifications. Dry tap is istics with other BM failure disorders.1–3 To reach the final unusual and suggests diagnoses other than AA. BM cellularity in diagnosis repeated marrow investigations are sometimes needed. AA, despite its definition is more often reduced rather than During the work out process, a number of diseases should be completely absent (Figure 1). Hypocellularity in AA has been set at considered in the differential diagnosis, namely: hypoplastic o30% hematopoietic cells. However, this definition has been myelodysplastic syndromes (MDS), hypoplastic AML and ALL, established mainly for children and young adults. In healthy large granular lymphocytosis, paroxysmal nocturnal hemo- elderly patients, BM cellularity is physiologically decreased.7 globinuria (PNH) and congenital marrow failure syndromes, such Therefore, cutoff o30% may not be applicable to elderly as Fanconi anemia and dyskeratosis congenita1 (Table 1). There patients. It is not rare to find aplastic subcortical areas in a are three diagnostic steps to define AA: (I) confirm the suspicion of biopsy of an otherwise healthy patient. Increase in stromal cells AA and exclude other BM failure diseases; (II) define the severity of such as plasma cells, lymphocytes sometimes forming follicles and the disease and (III) characterize the AA. mast cells are typical but confounding findings. Stromal cells hence have to be excluded in the global evaluation of marrow cellularity. Presence of ‘hot spots’ (Figure 1) with dominating CONFIRM THE SUSPICION OF AA AND EXCLUDE OTHER BM erythropoiesis and a certain degree of dyserythropoiesis is a FAILURE DISEASES typical feature in AA. Immunostaining has become mandatory for For the diagnosis of AA, the presence of pancytopenia and the topographic identification of various cell population, including proof of an empty BM without major dysplastic changes are increased blast cells, and might eventually identify the unusual mandatory. All evaluations needed for the diagnosis of AA are association with lymphoma.8 Flow cytometry of BM may listed in Table 2. AA is clinically characterized by no enlarged contribute to identifying abnormal populations. lymph nodes, no splenomegaly and no hepatomegaly. Pancyto- Cytogenetic investigations should be systematically performed penia is the main manifestation in the peripheral blood. Anemia is in AA patients. Cytogenetic abnormalities can be present in up to accompanied by reticulocytopenia, and macrocytosis is a common 12% of otherwise typical AA patients.9 Abnormal cytogenetic feature. The RBCs should not present relevant anisocytosis and clones often are small, may arise during the course of the poikilocytosis. Lymphocyte count is usually preserved. In early disease,10,11 or may be transient and disappear spontaneously/ stages isolated cytopenia, particularly thrombocytopenia can be after immunosuppression. An abnormal cytogenetic clone does seen. Monocytopenia can be present and imposes the differential not necessarily imply the diagnosis of MDS or AML. In marrow diagnosis of hairy cell leukemia. Careful examination of the blood failure syndromes, cytogenetic evaluation may be difficult because film is needed to exclude dysplastic neutrophils, the presence of of the lack of metaphases. In such a situation, FISH analysis erythroblasts, immature myeloid cells, abnormal platelets or other seeking for specific chromosomes aberrations is recommended. abnormal cells, such as hairy cells. Fetal Hb can be increased in Most frequent anomalies include trisomy 8, trisomy 6, 5q À , AA,4 in children pre-transfusional increase in fetal Hb imposes the anomalies of chromosome 7 and 13.

1Department of Hematology, University Hospital of Basel, Basel, Switzerland and 2Department of Hematology, G Gaslini Children’s Hospital, Genova, Italy. Correspondence: Dr A Rovo´ , Department of Hematology, University Hospital of Basel, Petersgraben 4, 4031 Basel, Switzerland. E-mail: [email protected] Received 11 October 2012; accepted 11 October 2012; published online 19 November 2012 Diagnosis in aplastic anemia A Rovo´ et al 163 CHARACTERIZE THE AA Table 1. Aplastic anemia, main differential diagnosis entities AA and PNH Acquired aplastic anemia There is a close correlation between AA and PNH.21,22 Patients Pure erythroaplasia with typical PNH can develop AA in the course of their disease and Pure white cell aplasia 23 Hypocellular refractory cytopenia of unknown significance patients with AA often present a PNH clone. Even the presence Hypoplastic MDS or acute leukemia of a very small PNH clone is a strong argument for a marrow Paroxysmal nocturnal hemoglobinuria failure syndrome. Flow cytometry is the Gold Standard method for T-LGL-leukemia with pancytopenia screening and diagnosis of PNH.24 This is currently best achieved by analysis of glycophosphatidylinositol (GPI)-linked Ags using Congenital marrow failure syndromes MoAbs and fluorescent aerolysin.25,26 About 40–50% of patients FA with acquired AA have a detectable PNH clone. Most clones are DKC small and patients do not have symptoms related to PNH. In some CAMT in aplastic phase DBA, SDS, congenital hypoplastic anemia patients, the PNH clone can increase after immunosuppressive treatment. In such case, the patient may present the typical Abbreviations: CAMT ¼ congenital amegakaryocytic thrombocytopenia; symptoms and complications of the disease.23 PNH clone size DBA ¼ Diamond Blackfan anemia; DKC ¼ dyskeratosis congenita; FA ¼ measurements should be performed at presentation and Fanconi anemia; LGL ¼ large granular lymphocytosis; MDS ¼ followed-up on serial monitoring every 6–12 months. myelodysplastic syndrome; SDS ¼ Shwachman Diamond syndrome.

AA and HLA-DR2/HLA-DRB1*15 The distinction between AA and hypoplastic MDS may be a HLA-DR typing might be useful for predicting a response to difficult diagnostic task. Both marrow failure syndromes show 27 12,13 immunosuppression in AA patients. HLA-DR2 and particularly markedly hypocellular BM with increased fat cells. The HLA-DRB1*15 (DRB1*1501 and DRB1*1502)28 have been absence of dysplasia particularly of the megakaryopoiesis and associated with some characteristics and to affect the outcome the absence of blast cells, as well as the lack of increased numbers of AA. Patients possessing HLA-DR15 tend to be older and actually of CD34 and CD117-positive cells by immunohistochemistry is the 450% of the patients with HLA-DRB1*1502 are older than 40 most conspicuous element supporting the diagnosis of AA. years of age. In Japanese patients, DRB1*1501 seems to be Nevertheless, in many cases changes in the karyotype, for associated with the presence of a small population of PNH-type instance, the presence of a monosomy 7, are the only criterion cells and a good response to the immunosuppressive therapy in favor of a hypoplastic MDS. Main characteristics of both entities (IST).29 In a recent study on 37 Korean patients with severe AA, are summarized in Table 3. In children, one of the most prominent responders to immunosuppressive treatment had a significantly features for identifying refractory cytopenia is the presence of higher HLA-DR15 and lower DR4 frequency compared with non- erythropoiesis with uni- or multifocal clustering of predominantly responders.30 The response rates in the best (DR15( þ )/DR4( À )), immature forms with increased mitoses, occasionally with atypia. intermediate, and poor response groups (DR15( À )/DR4( þ )) were The granulopoiesis is typically sparsely dispersed or lacking and 88.9%, 38.5% and 0%, respectively (P ¼ 0.00001). At the allelic megakaryopoiesis usually decreased with micromegakaryocytes 14 level, DRB1*1501 and closely linked DQB1*0602 were associated or other dysplastic changes. Despite similar clinical with a good response and DRB1*0405 and closely linked presentations, distinct cytokine profiles were observed between DQB1*0401 with a poor response to IST. AA and hypocellular MDS. Characteristic pattern of cytokines such as TPO and chemokine (C–C motif) ligand 3 might in future be useful tools to better discriminate both entities in clinical Hepatitis-associated AA 15 practice. Seronegative hepatitis is documented in 5–10% of patients with For the diagnosis of AA, a positive family history including other acquired AA.31–35 It typically occurs in young, healthy males with members affected with anemia, macrocytosis, cytopenia or severe but self-limited liver inflammation. A common inciting malignancies should invoke an inherited BM failure syndrome. A infectious cause could be involved both in the liver disease and in normal clinical examination does not definitively rule out a the BM failure.31 Indeed, in hepatitis-associated AA, similar skewed 16,17 ‘cryptic’ dyskeratosis congenita or a non-classical Fanconi T-cell repertoires have been detected in the liver and in the 18 anemia. The presence of unusual clinical features should alert peripheral blood lymphocytes.36 Patients with post-hepatitis AA the possibility of a congenital form of AA. Peripheral blood do not respond differently to immunosuppressive treatment lymphocytes should be tested for spontaneous and compared with patients with idiopathic acquired AA. diepoxybutane or mitomycin C-induced chromosomal breakage to identify or exclude Fanconi anemia. This should be performed in all marrow failure patients who are candidates to hematopoietic AA associated with other autoimmune disorders SCT. Screening should also include sibling donors of Fanconi Associations of AA with other autoimmune disease (AID) have anemia patients. Telomere shortening is a consistent and typical been shown in single-case reports.37–39 In a single-center report, finding of dyskeratosis congenita. Telomere length measurement 5.3% of the patients had an AID before the diagnosis of AA and of leukocytes from peripheral blood can be considered in any 4.5% of them developed an AID after diagnosis and treatment for marrow failure syndrome, but it does not belong yet to standard AA.40 AID can appear at any time before or after the AA. The screening. frequency of a concomitant AID seems higher in older AA patients as 425% of AA patients diagnosed after 50 years of age presented a concomitant AID. In a large multicenter study of the Severe Aplastic Anemia Working Party (SAAWP) of the European DEFINE THE SEVERITY OF THE DISEASE Group for Blood and Marrow Transplantation (EBMT), 50 of 1251 Once the diagnosis of AA has been established, the severity of the AA patients had an AID.41 Whether or not the immunosuppression disease has to be defined. The severity is based exclusively on applied to treat the AA has an influence on the outcome of the values of the peripheral blood. Accordingly, from Table 4, three AID still remains a controversial topic. In consideration of the groups of AA are defined7,19,20 severe AA, very severe AA and non- frequency of a concomitant AID in AA patients, it is unlikely that severe AA. both diseases appear together just by chance.

& 2013 Macmillan Publishers Limited Bone Marrow Transplantation (2013) 162 – 167 Diagnosis in aplastic anemia A Rovo´ et al 164 Table 2. Suggested diagnostic workup for AA

Diagnostic tool Provide information for Full blood count Diagnosis and severity criteria Reticulocyte count (automated or microscopic counting) Diagnosis and severity criteria Peripheral blood film examination Differential diagnosis Liver function test Seeking for hepatitis association Viral hepatitis studies (serological and DNA/RNA). RNA genome of Association with viral hepatitis RNA viruses such as HCV, HDV, HEV and HGV can be qualitatively detected by RT-PCR reaction and DNA of parvovirus B19 can be detected by Nested PCR. These studies along with EBV, CMV and other viral studies can be performed also on marrow BM aspirate for morphology, cytogenetics with optional FISH-analysis Diagnosis and differential diagnosis, prognosis (search for À 7; þ 8, 5q-, and so on), immunophenotyping and iron staining Trephine biopsy with CD34 and CD 117 immunostaining for blast Diagnosis, differential diagnosis, prognosis detection PNH clone with a sensitive multicolor flow cytometry. Evaluation Association with PNH clones. Diagnosis and differential diagnosis, samples from peripheral blood, searching clones in neutrophils, prognosis monocytes and erythrocyte population (erythrocyte If not transfused) Flow cytometry evaluating BM aspiration: searching for monoclonal Differential diagnosis myeloid neoplasms. Search for association with B- or T-cell population, blast population. Abnormal patterns of lymphoma maturation/differentiation as signs of dysplasia Autoantibody screen (panel according to clinical presentation). Association with an autoimmune disease Including anti-nuclear Ab and anti-DNA Ab. If systemic lupus erythematosus as underlying disease is suspected Investigation for mycobacterial infections (mainly atypical If infection is suspected. BM cultures/staining AAFB in histology mycobacterium, less frequent TBC) specimen. In vitro colony assay—results not well standardized. Not available in Growing patterns can be useful in the differential diagnosis between all centers marrow failures and MDS Vitamin B12, folic acid, iron status, fibrinogen Ruled out vitamin deficiency. Ferritin and fibrinogen can be useful if hemophagocytosis syndrome is suspected Serum bilirubin and LDH Not specific. Possibly increased because of minor degree of ineffective erythropoiesis

Screen for inherited disorders Positive chromosomal breakage by MMC or DEB test represents the In patients younger than 50 years if Fanconi anemia is suspected. current diagnostic golden standard for Fanconi anemia Candidates for HSCT (relevant data for conditioning regimens selection) Telomere length measurement As screening test when congenital forms are suspected. Mandatory if DKC suspected. Prognosis relevance in acquired forms? Mutation analysis of TERC/TERT. Rarely, with TINF2 gene mutation Differential diagnosis with hidden forms of autosomal dominant DKC 1–10% of all idiopathic forms are positive TINF2, NHP2, NOP10, DKC1 mutation When recognizable phenotype of DKC cMPL All patients with congenital amegakaryocytic thrombocytopenia have mutations in the TPO receptor gene c-Mpl. Useful differential diagnosis with aplastic anemia in the first years of life

Additional tests Chest X-ray To exclude infection Abdominal ultrasound scan and echocardiogram Enlarged spleen and/or lymph nodes (malignant hematological disease). Abnormal organ formation or anatomical displacement (Fanconi anemia). Differential diagnosis HLA-typing (search for HLA-DRB1*15) HLA-DR typing might be useful for predicting a response to immunosuppression in AA patients HLA-typing, family typing, eventually search for non related donor When HSCT is indicated Vertebral MRI Uniformly fatty marrow in AA vs a spotty mixture of hypo and hyper cellular marrow in MDS Confocal microscopy: tissues are immunostained and imaged in Only used in research. Three-dimensional images can be assessed three dimensions by confocal fluorescence and reflection microscopy qualitatively and quantitatively to appreciate the distribution of cell types and their interrelationships, with minimal perturbations of the tissue47

Abbreviations: AA ¼ aplastic anemia; AAFB ¼ acid alcohol fast bacilli; cMPL ¼ gene for the TPO receptor; DEB ¼ diepoxybutane; DKC ¼ dyskeratosis congenita; HCV ¼ hepatitis C virus; HDV ¼ hepatitis D virus; HEV ¼ hepatitis E virus; HGV ¼ hepatitis G virus; HSCT ¼ hematopoietic SCT; LDH ¼ lactate dehydrogenase; MDS ¼ myelodysplastic syndrome; MMC ¼ mytomycin c; MRI ¼ magnetic resonance imaging; PNH ¼ paroxysmal nocturnal hemoglobinuria; RT–PCR ¼ reverse transcriptase-PCR; TBC ¼ tuberculosis; TERC ¼ telomerase RNA component; TERT ¼ telomerase reverse transcriptase; TINF2 ¼ telomeric repeat binding protein 2.

RESPONSE CRITERIA OF AA X150 Â 109/L and Hb X120 g/L. A partial response (PR) includes, The definition of the response criteria to treatment belongs to the transfusion independency in patients who needed transfusions diagnostic tasks. The response criteria of AA depend in part on the before treatment, with either improvement of the severity degree severity of the disease before treatment. A complete response (CR) of the AA, or neutrophil counts increasing X0.5 Â 109/L and is defined in any case as a normalization of the blood values, platelet count 20X0 Â 109/L, in case the values were lower before which are neutrophil count X1.5 Â 109/L, platelet count treatment. Non-response is considered the persistence of

Bone Marrow Transplantation (2013) 162 – 167 & 2013 Macmillan Publishers Limited Diagnosis in aplastic anemia A Rovo´ et al 165

Figure 1. BM histology: aplastic BM histology (a), and hypocellular bone marrow with ‘hot spots’ (b) from a patient with severe AA. (Picture courtesy of Professor Stephan Dirnhofer, Institute of Pathology, University Hospital of Basel, Basel, Switzerland.)

Table 3. Main diagnostic characteristics of aplastic anemia and hypoplastic myelodysplastic syndromes

Aplastic anemia Hypoplastic MDS

Cytopenia Present Present BM cellularity Aplastic (o10% cellularity ) or hypocellular Hypocellular BM hematopoiesis Erythropoiesis Present in nest, ‘hot spots’ Present Myelopoiesis Typically decreased Present Megakaryopoiesis Decreased or absent Present

Dysplasia Erythropoiesis Possible Possible Myelopoiesis Normal morphology Possible Megakaryopoiesis Normal morphology Possible

Blasts Absent Variable CD34 þ or CD117 þ immunohistochemistry Nearly absent Normal or increased Marrow fibrosis Absent Possible Karyotype Clonal abnormality possible (about 12%) À 7/del(7q) À 5/del(5q) PNH clone Frequent Unusual Splenomegaly at diagnosis Absent Possible

Abbreviations: MDS ¼ myelodysplastic syndrome; PNH ¼ paroxysmal nocturnal hemoglobinuria.

transfusion dependency or of values lower than those mentioned children, lower WBC count (o2.0 Â 109/L) was the most above. The definition of PR in AA is not fully satisfactory because it significant predictive marker of better response.43 In patients includes patients with very good and stable PR and other patients receiving G-CSF, the lack of a neutrophil response (o0.5 Â 109/L) who have barely obtained values above the minimal criteria. by day 30 was associated with significantly lower response rate and survival.44 Patients refractory to two consecutive courses of anti-thymocyte globulin (ATG) have a very low chance to respond FUTURE DIRECTIONS IN THE DIAGNOSIS OF ACQUIRED AA to a third course of anti-thymocyte globulin, and may be suitable Since the introduction of the concept of AA by Paul Ehrlich in 1888 candidates for novel therapeutic options.45 and despite the current better understanding of the underlying A recent publication showed that telomere shortening in AA mechanisms involved in this disease a clear delineation among BM patients was associated with both numerical and structural failures is still a matter of debate. The diagnostic challenge for the chromosome abnormalities. Patients with shorter telomeres were forthcoming years is to identify AA patients at risk of failing IST, at higher risk of malignant transformation. Shorter average and therefore being candidates for early hematopoietic SCT, and to telomere lengths inversely correlated with monosomy 7 at improve diagnostic standardization of inherited BM failures. During diagnosis (Figure 2).46 This result might have clinical implications the past years, a number of studies focused on parameters to because affected individuals may benefit from therapies that predict response after IST but were not able to define refractori- eventually eliminate the shortest (and dysfunctional) telomeres. ness. Baseline absolute reticulocyte and lymphocyte counts were A measurement of telomere length arises indeed as an interesting defined as simple predictors of response following IST.42 In prognostic tool in acquired AA.

& 2013 Macmillan Publishers Limited Bone Marrow Transplantation (2013) 162 – 167 Diagnosis in aplastic anemia A Rovo´ et al 166 Table 4. Definition of disease severity based on peripheral values 5 Hasle H, Baumann I, Bergstrasser E, Fenu S, Fischer A, Kardos G et al. The Inter- and BM findings national Prognostic Scoring System (IPSS) for childhood myelodysplastic syn- drome (MDS) and juvenile myelomonocytic leukemia (JMML). Leukemia 2004; 18: 2008–2014. SAA19 6 Hasle H. Myelodysplastic and myeloproliferative disorders in children. Curr Opin At least two of the following three criteria have to be fulfilled: Pediatr 2007; 19: 1–8. Reticulocytes o60 Â 109/L (using an automated analyzer) or 7 Tuzuner N, Bennett JM. Reference standards for bone marrow cellularity. Leuk Res o 20 Â 109/l (manual count)a 1994; 18: 645–647. Platelets o 20 Â 109/L 8 Medinger M, Buser A, Stern M, Heim D, Halter J, Rovo A et al. Aplastic anemia in Neutrophil count o0.5 Â 109/L association with a lymphoproliferative neoplasm: coincidence or causality? Leuk vSAA20 Res 2012; 36: 250–251. Same criteria of SAA have to be fulfilled; but the neutrophil count 9 Gupta V, Brooker C, Tooze JA, Yi QL, Sage D, Turner D et al. Clinical relevance of cytogenetic abnormalities at diagnosis of acquired aplastic anaemia in adults. has to be o 0.2 Â 109/L Br J Haematol 2006; 134: 95–99. Non-severe AA 10 Socie G, Rosenfeld S, Frickhofen N, Gluckman E, Tichelli A. Late clonal diseases of Patients not fulfilling the criteria for SAA and vSAA treated aplastic anemia. Semin Hematol 2000; 37: 91–101. 11 Tichelli A, Gratwohl A, Nissen C, Speck B. Late clonal complications in severe Abbreviations: AA ¼ aplastic anemia; SAA ¼ severe AA; vSAA ¼ very severe aplastic anemia. Leuk Lymphoma 1994; 12: 167–175. AA. aThe different values are because automated count may over-estimate 12 Bennett JM, Orazi A. Diagnostic criteria to distinguish hypocellular acute myeloid the counting at low level of reticulocyte counts, that is, it reads 50 Â 109/L leukemia from hypocellular myelodysplastic syndromes and aplastic anemia: but in reality they are less. recommendations for a standardized approach. Haematologica 2009; 94:264–268. 13 Barrett J, Saunthararajah Y, Molldrem J. Myelodysplastic syndrome and aplastic anemia: distinct entities or diseases linked by a common pathophysiology? 0.6 Semin. Hematol 2000; 37: 15–29. Telomere 1st Quartile; 4-yr evolution = 18.8% 14 Cantu RA, Fenu S, Kerndrup G, van Wering ER, Niemeyer CM, Baumann I. Eva- Telomere 2nd Quartile; 4-yr evolution = 8.3% luation of dysplastic features in myelodysplastic syndromes: experience from the morphology group of the European Working Group of MDS in Childhood Telomere 3rd Quartile; 4-yr evolution = 2.5% th (EWOG-MDS). Ann Hematol 2005; 84: 429–433. 0.4 Telomere 4 Quartile; 4-yr evolution = 2.5% 15 Feng X, Scheinberg P, Wu CO, Samsel L, Nunez O, Prince C et al. Cytokine sig- nature profiles in acquired aplastic anemia and myelodysplastic syndromes. Haematologica 2011; 96: 602–606. P = 0.018 16 Walne AJ, Dokal I. Dyskeratosis congenita: a historical perspective. Mech Ageing Dev 2008; 129: 48–59. 0.2 17 Walne AJ, Dokal I. Advances in the understanding of dyskeratosis congenita. Br J Haematol 2009; 145: 164–172. 18 Auerbach AD. Fanconi anemia and its diagnosis. Mutat Res 2009; 668: 4–10. Probability of evolution Probability 19 Camitta BM, Rappeport JM, Parkman R, Nathan DG. Selection of patients for bone marrow transplantation in severe aplastic anemia. Blood 1975; 45: 355–363. 0.0 20 Bacigalupo A, Hows J, Gluckman E, Nissen C, Marsh J, Van Lint MT et al. Bone marrow transplantation (BMT) versus immunosuppression for the treatment of 01234 5 6 severe aplastic anaemia (SAA): a report of the EBMT SAA working party. Years Br J Haematol 1988; 70: 177–182. 21 Young NS, Maciejewski JP, Sloand E, Chen G, Zeng W, Risitano A et al. Figure 2. Telomere length in AA patients. Probability of evolution to The relationship of aplastic anemia and PNH. Int. J Hematol 2002; 76(Suppl 2): MDS and/or acute leukemia in patients with acquired AA and 168–172. treated with immunosuppression, according to pre-treatment age- 22 Pu JJ, Brodsky RA. Paroxysmal nocturnal hemoglobinuria from bench to bedside. adjusted telomere length. Patients were divided into quartiles based Clin Transl Sci 2011; 4: 219–224. on age-adjusted telomere length (first quartile, shorter telomeres; 23 Pu JJ, Mukhina G, Wang H, Savage WJ, Brodsky RA. Natural history of paroxysmal and fourth quartile, longer telomeres). Patients in the shorter nocturnal hemoglobinuria clones in patients presenting as aplastic anemia. Eur J quartile (first) had a higher chance to evolve to myelodysplasia and/ Haematol 2011; 87: 37–45. or leukemia (24.5%) at 5 years in comparison with patients in the 24 Hall SE, Rosse WF. The use of monoclonal antibodies and flow cytometry in the second (10.7%), third (4.8%) and fourth (9.7%) quartiles (log-rank, 1 46 diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 1996; 87: 5332–5340. P40.06). Modified from Calado et al. 25 Brodsky RA, Mukhina GL, Li S, Nelson KL, Chiurazzi PL, Buckley JT et al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin. Am J Clin Pathol 2000; 114: 459–466. CONFLICT OF INTEREST 26 Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglo- The authors declare no conflict of interest. binuria and related disorders by flow cytometry. Cytometry B Clin Cytom 2010; 78: 211–230. ACKNOWLEDGEMENTS 27 Kapustin SI, Popova TI, Lyschov AA, Togo AV, Abdulkadyrov KM, Blinov MN. HLA- DR2 frequency increase in severe aplastic anemia patients is mainly attributed to ERG spa, SAAR Depositi Oleari Portuali, Rimorchiatori Riuniti and Cambiaso and Risso the prevalence of DR15 subtype. Pathol Oncol Res 1997; 3: 106–108. are acknowledged for their support to the activity of the Hematology Unit of G 28 Sugimori C, Yamazaki H, Feng X, Mochizuki K, Kondo Y, Takami A et al. Roles of Gaslini Children’s Hospital, Genova, Italy. DRB1 *1501 and DRB1 *1502 in the pathogenesis of aplastic anemia. Exp Hematol 2007; 35: 13–20. 29 Yoshida N, Yagasaki H, Takahashi Y, Yamamoto T, Liang J, Wang Y et al. Clinical REFERENCES impact of HLA-DR15, a minor population of paroxysmal nocturnal haemoglobi- 1 Young NS, Calado RT, Scheinberg P. Current concepts in the pathophysiology and nuria-type cells, and an aplastic anaemia-associated autoantibody in children with treatment of aplastic anemia. Blood 2006; 108: 2509–2519. acquired aplastic anaemia. Br J Haematol 2008; 142: 427–435. 2 Shimamura A. Clinical approach to marrow failure. Hematology Am Soc Hematol 30 Song EY, Kang HJ, Shin HY, Ahn HS, Kim I, Yoon SS et al. Association of human Educ Program 2009; 329–337. leukocyte antigen class II alleles with response to immunosuppressive therapy in 3 Young NS. Acquired aplastic anemia. Ann Intern Med 2002; 136: 534–546. Korean aplastic anemia patients. Hum Immunol 2010; 71: 88–92. 4 Tichelli A, Gratwohl A, Nissen C, Signer E, Stebler GC, Speck B. Morphology in 31 Brown KE, Tisdale J, Barrett AJ, Dunbar CE, Young NS. Hepatitis-associated aplastic patients with severe aplastic anemia treated with antilymphocyte globulin. Blood anemia. N Engl J Med 1997; 336: 1059–1064. 1992; 80: 337–345. 32 Young NS. Flaviviruses and bone marrow failure. JAMA 1990; 263: 3065–3068.

Bone Marrow Transplantation (2013) 162 – 167 & 2013 Macmillan Publishers Limited Diagnosis in aplastic anemia A Rovo´ et al 167 33 Locasciulli A, Bacigalupo A, Bruno B, Montante B, Marsh J, Tichelli A et al. Hepa- with aplastic anemia reported to the registry of the European group for blood and titis-associated aplastic anaemia: epidemiology and treatment results obtained in marrow transplantation. Acta Haematol 2010; 124: 19–22. Europe. A report of The EBMT aplastic anaemia working party. Br J Haematol 2010; 42 Scheinberg P, Wu CO, Nunez O, Young NS. Predicting response to immunosup- 149: 890–895. pressive therapy and survival in severe aplastic anaemia. Br J Haematol 2009; 144: 34 Mary JY, Baumelou E, Guiguet M. Epidemiology of aplastic anemia in France: a 206–216. prospective multicentric study. The French Cooperative Group for Epidemiolo- 43 Yoshida N, Yagasaki H, Hama A, Takahashi Y, Kosaka Y, Kobayashi R et al. Pre- gical Study of Aplastic Anemia. Blood 1990; 75: 1646–1653. dicting response to immunosuppressive therapy in childhood aplastic anemia. 35 Rauff B, Idrees M, Shah SA, Butt S, Butt AM, Ali L et al. Hepatitis associated aplastic Haematologica 2011; 96: 771–774. anemia: a review. Virol J 2011; 8: 87. 44 Tichelli A, Schrezenmeier H, Socie G, Marsh J, Bacigalupo A, Duhrsen U et al. A 36 Lu J, Basu A, Melenhorst JJ, Young NS, Brown KE. Analysis of T-cell repertoire in randomized controlled study in patients with newly diagnosed severe aplastic hepatitis-associated aplastic anemia. Blood 2004; 103: 4588–4593. anemia receiving antithymocyte globulin (ATG), cyclosporine, with or without 37 Grey-Davies E, Hows JM, Marsh JC. Aplastic anaemia in association with coeliac G-CSF: a study of the SAA Working Party of the European Group for Blood and disease: a series of three cases. Br J Haematol 2008; 143: 258–260. Marrow Transplantation. Blood 2011; 117: 4434–4441. 38 Antic M, Lautenschlager S, Itin PH. Eosinophilic fasciitis 30 years after - what do 45 Gupta V, Gordon-Smith EC, Cook G, Parker A, Duguid JK, Wilson KM et al. A third we really know? Report of 11 patients and review of the literature. Dermatology course of anti-thymocyte globulin in aplastic anaemia is only beneficial in pre- 2006; 213: 93–101. vious responders. Br J Haematol 2005; 129: 110–117. 39 Hinterberger-Fischer M, Kier P, Forstinger I, Lechner K, Kornek G, Breyer S et al. 46 Calado RT, Cooper JN, Padilla-Nash HM, Sloand EM, Wu CO, Scheinberg P et al. Coincidence of severe aplastic anaemia with multiple sclerosis or thyroid dis- Short telomeres result in chromosomal instability in hematopoietic cells orders. Report of 5 cases. Acta Haematol 1994; 92: 136–139. and precede malignant evolution in human aplastic anemia. Leukemia 2012; 26: 40 Stalder MP, Rovo A, Halter J, Heim D, Silzle T, Passweg J et al. Aplastic anemia and 700–707. concomitant autoimmune diseases. Ann Hematol 2009; 88: 659–665. 47 Takaku T, Malide D, Chen J, Calado RT, Kajigaya S, Young NS. Hematopoiesis in 3 41 Cesaro S, Marsh J, Tridello G, Rovo A, Maury S, Montante B et al. Retrospective dimensions: human and murine bone marrow architecture visualized by confocal survey on the prevalence and outcome of prior autoimmune diseases in patients microscopy. Blood 2010; 116: e41–e55.

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